Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging

Background In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177. Results Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h. Conclusions This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy

From defined polymer architectures to structure-property relationships in vivo

Makromolekulare Wirkstoffträgersysteme sind von starkem Interesse bezüglich der klinischen Anwendung chemotherapeutischer Agenzien. Um ihr klinisches Potential zu untersuchen ist es von besonderer Bedeutung das pharmakokinetische Profil in vivo zu bestimmen. Jede Veränderung der Polymerstruktur beeinflusst die Körperverteilung des entsprechenden Makromoleküls. Aufgrund dessen benötigt man detailliertes Wissen über Struktur-Eigenschaftsbeziehungen im lebenden Organismus, um das Nanocarrier System für zukünftige Anwendungen einzustellen. In dieser Beziehung stellt das präklinische Screening mittels radioaktiver Markierung und Positronen-Emissions-Tomographie eine nützliche Methode für schnelle sowie quantitative Beobachtung von Wirkstoffträgerkandidaten dar. Insbesondere poly(HPMA) und PEG sind im Arbeitsgebiet Polymer-basierter Therapeutika stark verbreitet und von ihnen abgeleitete Strukturen könnten neue Generationen in diesem Forschungsbereich bieten.rnDie vorliegende Arbeit beschreibt die erfolgreiche Synthese verschiedener HPMA und PEG basierter Polymer-Architekturen – Homopolymere, Statistische und Block copolymere – die mittels RAFT und Reaktivesterchemie durchgeführt wurde. Des Weiteren wurden die genannten Polymere mit Fluor-18 und Iod-131 radioaktiv markiert und mit Hilfe von microPET und ex vivo Biodistributionsstudien in tumortragenden Ratten biologisch evaluiert. Die Variation in Polymer-Architektur und darauffolgende Analyse in vivo resultierte in wichtige Schlussfolgerungen. Das hydrophile / lipophile Gleichgewicht hatte einen bedeutenden Einfluss auf das pharmakokinetische Profil, mit besten in vivo Eigenschaften (geringe Aufnahme in Leber und Milz sowie verlängerte Blutzirkulationszeit) für statistische HPMA-LMA copolymere mit steigendem hydrophoben Anteil. Außerdem zeigten Langzeitstudien mit Iod-131 eine verstärkte Retention von hochmolekularen, HPMA basierten statistischen Copolymeren im Tumorgewebe. Diese Beobachtung bestätigte den bekannten EPR-Effekt. Hinzukommend stellen Überstrukturbildung und damit Polymergröße Schlüsselfaktoren für effizientes Tumor-Targeting dar, da Polymerstrukturen über 200 nm in Durchmesser schnell vom MPS erkannt und vom Blutkreislauf eliminiert werden. Aufgrund dessen wurden die hier synthetisierten HPMA Block copolymere mit PEG Seitengruppen chemisch modifiziert, um eine Verminderung in Größe sowie eine Reduktion in Blutausscheidung zu induzieren. Dieser Ansatz führte zu einer erhöhten Tumoranreicherung im Walker 256 Karzinom Modell. Generell wird die Körperverteilung von HPMA und PEG basierten Polymeren stark durch die Polymer-Architektur sowie das Molekulargewicht beeinflusst. Außerdem hängt ihre Effizienz hinsichtlich Tumorbehandlung deutlich von den individuellen Charakteristika des einzelnen Tumors ab. Aufgrund dieser Beobachtungen betont die hier vorgestellte Dissertation die Notwendigkeit einer detaillierten Polymer-Charakterisierung, kombiniert mit präklinischem Screening, um polymere Wirkstoffträgersysteme für individualisierte Patienten-Therapie in der Zukunft maßzuschneidern.rnMacromolecular based drug delivery systems are of emerging interest regarding the clinical administration of (chemo) therapeutic agents. In order to investigate their clinical potential, it is crucial to determine the pharmacokinetic profile in vivo. Each alteration in polymer structure is influencing the body distribution of the respective macromolecule and thus deepening knowledge about structure-property relationships in the living organism is needed to adjust the nanocarrier system for future applications. In this regard, pre-clinical screening via radiolabeling combined with Positron Emission Tomography (PET) constitutes a useful tool for fast and quantitative monitoring of drug delivery candidates in vivo. Particularly p(HPMA) and PEG are widely exploited in the field of polymer based therapeutics and thus derivatized structures might provide new generations in this research area.rnThis work describes the successful synthesis of diverse HPMA and PEG based polymer architectures - including homopolymers, random and block copolymers – accomplished via RAFT polymerization and reactive ester chemistry. Furthermore, their radioactively labeling and biological evaluation was carried out by means of 18F and 131I combined with µPET imaging and ex vivo biodistribution in tumor bearing rats. The variation in polymer architecture and subsequent analysis in vivo resulted in some major conclusions. First, the hydrophilic / lipophilic balance had a major influence on the pharmacokinetic profile, demonstrating most favorable in vivo characteristics (low hepatic and spleenic uptake as well as prolonged blood circulation times) for HPMA-ran-LMA copolymers with increasing content of hydrophobic moiety. Second, long term biodistribution studies with iodine-131 demonstrated enhanced retention in the tumor tissue for high molecular weight HPMA based random copolymers thus proofing the well-known EPR-effect. Third, superstructure formation and hence polymer size are key factors for efficient tumor targeting since polymer structures above 200 nm in diameter are rapidly recognized by the MPS and cleared from the bloodstream. In this regard, the herein synthesized HPMA block copolymers were chemically modified with PEG side chains to induce a decrease in size as well as reduced blood clearance, yielding enhanced tumor accumulation in the Walker 256 mammary carcinoma model. Forth, the overall polymer architecture as well as molecular weight are strongly influencing the body distribution of HPMA / PEG based polymers and their efficacy of tumor treatment is significantly depending on the properties of each individual tumor. Due to this observation, the present work is underlining the necessity of a precise polymer characterization in combination with pre-clinical screening to tailor polymeric carrier systems for individualized patients’ (chemo) therapy in the future.r

A Minimal Hydrophobicity Is Needed To Employ Amphiphilic p(HPMA)-co-p(LMA) Random Copolymers in Membrane Research

Because a polymer environment might be milder than a detergent micelle, amphiphilic polymers have attracted attention as alternatives to detergents in membrane biochemistry. The polymer poly­[<i>N</i>-(2-hydroxypropyl)-methacrylamid] [p­(HPMA)] has recently been modified with hydrophobic lauryl methacrylate (LMA) moieties, resulting in the synthesis of amphiphilic p­(HPMA)-co-p­(LMA) polymers. p­(HPMA)-co-p­(LMA) polymers with a LMA content of 5 or 15% have unstable hydrophobic cores. This, on one hand, promotes interactions of the hydrophobic LMA moieties with membranes, resulting in membrane rupture, but at the same time prevents formation of a hydrophobic, membrane mimetic environment that is sufficiently stable for the incorporation of transmembrane proteins. On the other hand, the p­(HPMA)-co-p­(LMA) polymer with a LMA content of 25% forms a stable hydrophobic core structure, which prevents hydrophobic interactions with membrane lipids but allows stable incorporation of membrane proteins. On the basis of our data, it becomes obvious that amphiphilic polymers have to have threshold hydrophobicities should an application in membrane protein research be anticipated

Additional file 1: of Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging

Supporting information. (ZIP 77 kb

HPMA-LMA Copolymer Drug Carriers in Oncology: An in Vivo PET Study to Assess the Tumor Line-Specific Polymer Uptake and Body Distribution

Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with ¹⁸F and organ/tumor uptake was followed by μPET imaging and <i>ex vivo</i> biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy

HPMA-LMA Copolymer Drug Carriers in Oncology: An in Vivo PET Study to Assess the Tumor Line-Specific Polymer Uptake and Body Distribution

Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with ¹⁸F and organ/tumor uptake was followed by μPET imaging and <i>ex vivo</i> biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy